This case claims priority to U.S. Provisional Patent Application No. 60/294,405 filed Jun. 7, 2000 now abandoned and entitled “DNA Encoding Human Voltage Gated Sodium Channel β1A Subunit” and U.S. Provisional Patent Application No. 60/236,664 filed Sep. 29, 2000 and entitled “DNA Encoding Human Voltage Gated Sodium Channel β1A Subunit.”
Rapid entry of sodium ion into cells causes depolarization and generation of the action potential. Such entry of sodium ions in response to voltage change on the plasma membrane in excitable cells is mediated by voltage gated sodium channels (VGSC). Therefore, voltage gated sodium channels play a fundamental role in the control of neuronal excitability in the central and peripheral nervous systems. The VGSC is a protein complex comprising at least a large (200-300 kDa), pore forming, α subunit and two small (30-40 kD) regulatory β1 and β2 subunits (Catterall, 1992 & 1993; Isom et al., 1992; Isom et al., 1995). It is well known that VGSC α subunits determine the basic properties of the channel because expression of the a subunit of VGSC alone in the heterologous expression systems such as HEK cells, CHL cells and Xenopus oocytes is sufficient to synthesize a functional, but altered, sodium channel. Co-expression of VGSC β1 and β2 subunits with the a subunit will usually normalize the channel properties in heterologous expression systems. In other words, VGSC β1 and β2 subunits modulate almost all the aspects of the channel properties including voltage dependent gating, activation and inactivation, and increase the number of functional channels on the plasma membrane. The VGSC β subunits may also be the rate limiting step controlling the increased expression of sodium channels (Caterall, 1992; Isom et al., 1994)
Molecular cloning studies have demonstrated that there are many different types of VGSC α subunits, which can be categorized based on their sensitivity to neurotoxin and tetrodotoxin (TTX) (Marban, et al. 1998). Because brain VGSC type I, IIA, III, skeletal muscle type I, sodium channel protein 6 (SCP6), its closely homologous peripheral nerve 4 (PN4), and peripheral nerve 1 (PN1) are blocked by TTX at nanomolar concentrations, they are termed TTX-sensitive (TTX-S) sodium channels. The cardiac sodium channel (H1), and peripheral nerve 3 (PN3/SNS) and NaN/SNS2 are normally blocked by TTX in the micromolar range, and are termed TTX-resistant (TTX-R) sodium channels.
The studies of VGSC β subunits are far behind those of VGSC α subunits. So far, only two types of β subunits, β1 and β2 (Isom, et al. 1992, 1994 & 1995) have been cloned and characterized. Recently, a novel VGSC β1A subunit, a splicing variant of the β1 subunit, has been identified from rat (Kazen-Gillespie, et al. 2000). Rat VGSC β1A subunit results from an apparent intron retention event. Analysis of rat genomic DNA indicated the divergent region (carboxyl region) of β1A is encoded by intron 3 with an in-frame termination codon. Like the VGSC β1 subunit, the β1A subunit increases sodium current density and [3H]Saxitoxin binding sites, and modulates voltage dependent activation and inactivation of the type IIA of VGSC. More interestingly, the expression level and pattern of the VGSC β1A in dorsal root ganglia (DRG) are changed significantly in the Chung animal neuropathic pain model (see below), which is consistent with the observation that the sodium current is increased after nerve injury. Both VGSC β1 and β1A subunits are integral membrane glycoproteins (Isom, et al. 1992, Kazen-Gillespie, et al. 2000) containing a single transmembrane domain at the carboxyl terminus and an extracellular amino-terminal immunoglobulin-like fold motif maintained by a single putative disulfide bridge between two highly conserved cysteine residues. VGSC β1 and β1A can be classified as members of the V-set of the Ig superfamily, which includes many cell adhesion molecule, suggesting that β1 and β1A subunits play roles not only in modulating sodium channel properties, but also in protein targeting and cell adhesion (Isom and Catteral, 1996).
An increase in the rate of spontaneous firing in neurons is often observed in peripheral sensory ganglia following nerve injury (Ochoa and Torebjork, 1980; Nordin et al., 1984; Devor, 1994; Woolf, 1994). It has been suggested that this hyperexcitability in neurons is due to altered sodium channel expression in some chronic pain syndromes (Tanaka et al., 1998). Increased numbers of sodium channels leading to inappropriate, repetitive firing of the neurons have been reported in the tips of injured axons in various peripheral nervous tissues such as the DRG which relay signals from the peripheral receptors into the central nervous system (Waxman and Brill, 1978; Devor et al., 1989; Matzner and Devor, 1992; Devor et al., 1992; England et al., 1994; Matzner and Devor, 1994; England et al., 1996). Transcripts encoding the α-III subunit, which are present at only very low levels in control DRG neurons, are expressed at moderate to high levels in axotomized DRG neurons together with elevated levels of α-I and α-II mRNAs (Waxman et al, 1994). Conversely, transcripts of sodium channel α-SNS are down-regulated in DRG neurons following axotomy (Dib-Hajj et al., 1996). Furthermore, the partial efficacy of sodium blocking agents is well documented in patients treated for neuropathic pain (Chabel et al., 1989; Devor et al., 1992; Omana-Zapata et al., 1997; Rizzo, 1997), providing an important link between increased sodium channel expression and neuropathic pain. Therefore, alterations in sodium channel expression and subsequent function may be a key molecular event underlying the pathophysiology of pain after peripheral nerve injury.
Recently the VGSC β1A subunit had been cloned and was reported to increase sodium current density at the plasma membrane when co-expressed with αIIA subunits in CHL fibroblasts (Kazan-Gillespie et al., 2000). β1A is developmentally regulated in the brain, but its potential role in neuropathic pain has not been previously explored. Therefore, the expression of β1A protein in DRG neurons using the Chung model of neuropathic pain (Kim and Chung, 1992) was investigated using a polyclonal antibody directed against a unique extracellular region of β1A not present in β1. Immunohistochemistry and computer-assisted image analysis documented significant up-regulation of VGSC β1A and β1 subunits following neuronal injury, compared to very low levels in the DRG from sham operated animal. The distinct punctate and membrane labeling distribution of β1A following peripheral nerve injury suggested active translation and possible accumulation into the plasma membrane, unlike β1, where the subcellular distribution remained diffuse.
To further explore the functions of VGSC β1A subunit, human VGSC β1A subunit has been cloned and characterized in this invention.